Comparison Evans Blue injection routes: Intravenous versus intraperitoneal, for measurement of blood–brain barrier in a mice hemorrhage model
Introduction
The blood–brain barrier (BBB) is a specialized vascular system consisting of endothelial cell tight junctions, basal lamina and glial processes (Rubin and Staddon, 1999). It separates circulating blood from cerebrospinal fluid in the central nervous system and has a low permeability to ionized water-soluble molecules with a molecular mass greater than 180 Da, unless the molecule can cross into the brain via specific active transporters (Kroll and Neuwelt, 1998). Disruption of the BBB following brain injury results in the development of vasogenic brain edema, a most life threatening event after such events as ICH (Xi et al., 2006). Since preservation of the BBB is a common goal among neuroprotective therapies, an objective method for evaluating blood brain barrier disruption is needed.
A common technique of evaluation involves the staining of plasma serum albumin, known as the Evans Blue assay. The major characteristic of Evans Blue stain is its ability to bind to serum albumin immediately after stain injection into the blood stream (Reeve, 1965). Since plasma albumin does not pass the BBB under normal physiologic conditions, spectrophotometric determination of Evans Blue stain accumulation is an easy and reliable way to estimate BBB permeability. In this study we compared different protocols of this assay and evaluated the difference in the Evans Blue stain accumulation in brain of mice after intraperitoneal versus intravenous injection of dry stain, at three different points of circulation time.
Section snippets
Animal groups and treatment methods
This study was conducted in accordance with the National Institutes of Health guidelines for the treatment of animals and was approved by the Animal Care and Use Committee at Loma Linda University. Male CD-1 mice (34–43 g, Charles River, MA, USA) were housed with a 12-h light/dark cycle with access to water and food ad libitum. A total of 58 mice were used. Animals were divided into two groups: a sham (needle-trauma only) group (6 mice) and an ICH group (42 mice). All animals received an
Results
No neurological deficit between ICH animals in different groups was observed.
Results of neurological testing are summarized in Fig. 1A–C. All animals that received collagenase injections demonstrated significant neurological deficit (p < 0.05 vs. sham). No statistically significant difference between experimental groups was observed.
Discussion
The aim of this study was to establish whether the administration route or circulation time would affect the amount of Evans Blue delivered across the blood–brain-barrier into the brain of mice after ICH.
Brain edema is most devastating life-threatening complication after ICH. Multiple forms of edema are present after ICH, but the main form is likely vasogenic (Xi et al., 2006, Adeoye et al., 2010) in origin. Several mechanisms have been proposed to be responsible for the development of
Conclusion
Evans Blue stain accumulation in the brains of mice after ICH is independent from administration route and circulation time.
Acknowledgment
We would like to thank Suzzanne Marcantonio (the Department of Anesthesiology, Loma Linda University) for excellent technical assistance.
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